GSM7658842: B cell, PT-sorted, BCR; Homo sapiens; RNA-Seq
Cryopreserved PBMCs from DTP-vaccinated donors were thawed quickly and resuspend in FACS buffer (2% FBS in PBS). Cells were stained by cell surface markers to obtain the antigen-experienced cell population (CD19+CD27+), Human Total Seq C Hashtags to retain the donor information, and one of the fluorescent-labeled toxins (DT, TT, PT) to obtain toxin-binding and non binding B cells. Sorting was done using BD Aria II Cell Sorter. Totally we have four groups of samples: DT-, TT-, PT-sorted B cells, and unsorted B cells, each of which are containing 4 donors origin. Library construction was performed according to the manufacturer's protocol (Single cell immune profiling, 5' v2, 10X Genomics). Hashtag-labeled B cells were resuspended in the master mix and loaded onto the chip together with partitioning oil and gel beads to generate GEM. The poly-A RNA from cell lysate was retrotranscripted to cDNA in each GEM containing the Illumina R1 sequencing primer, UMI, and 10x barcode. Simultaneously in the same partition, 10x barcoded DNA from the cell surface protein Feature barcode (hashtag) were produced. The pooled barcoded cDNA and DNA was then cleaned up, amplified by PCR, and separated by size to generate V(D)J and 5' Gene expression libraries, and cell surface protein (hashtag) libraries. During the library construction, Illumina R2 sequencing primer and sample index were added and finally, the libraries were sequenced.